: Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require diagnostic tools that are appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that is highly specific for the Puerto Rican Zika virus isolate, PRVABC59, and related isolates in the Asian clade. The assay does not detect Senegalese or Ugandan Zika isolates, or dengue, yellow fever, West Nile, or chikungunya viruses. The conditions described for the PRVABC59-LAMP assay allow direct detection of virus in infected cells, mosquitos, serum and blood without reverse transcription or RNA isolation. Time to detection of a single infectious particle in blood is 60 minutes in laboratory or field settings. It offers rapid, specific, sensitive and inexpensive detection of the Zika viruses currently circulating in the western hemisphere.